MARCO is an IFN-restricted immunometabolic decoy LPS receptor

MARCO is an IFN-restricted immunometabolic decoy LPS receptor | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results MARCO is an IFN-restricted immunometabolic decoy LPS receptor Shriram Ramani , Sara Cahill , Matthew Finnegan , Laurel Stine , Liraz Shmuel-Galia , View ORCID Profile Fiachra Humphries doi: https://doi.org/10.1101/2025.03.10.642106 Shriram Ramani 1 Division of Innate Immunity, Department of Medicine, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sara Cahill 1 Division of Innate Immunity, Department of Medicine, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Matthew Finnegan 1 Division of Innate Immunity, Department of Medicine, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Laurel Stine 1 Division of Innate Immunity, Department of Medicine, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Liraz Shmuel-Galia 2 Department of Pathology, University of Texas Southwestern Medical Centre , Dallas, Texas, USA 3 Department of Immunology, University of Texas Southwestern Medical Centre , Dallas, Texas, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Fiachra Humphries 1 Division of Innate Immunity, Department of Medicine, University of Massachusetts Chan Medical School , Worcester, MA 01605, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fiachra Humphries For correspondence: fiachra.humphries{at}umassmed.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Intracellular sensing of lipopolysaccharide is an essential component of pathogen detection that governs the innate immune response. However, how this process is controlled to maintain homeostasis and resolve inflammation is unclear. Here we show that MARCO is a macrophage decoy LPS sensor crucial for restraining caspase 11 activity and the non-canonical inflammasome. Remarkably, MARCO expression is controlled by the metabolite itaconate and the transcription factor NRF2. In the presence of IFN, itaconate mediated NRF2 stabilization is impaired, thus inhibiting MARCO expression and licensing optimal activation of the non-canonical inflammasome. Loss of MARCO augments non-canonical inflammasome activation and sensitized mice to septic shock. Together, this study identifies MARCO as a previously unknown LPS sensor and reveals an intricate immunometabolic homeostatic switch that allows for optimal immune responses and resolution of inflammation. Main Cytosolic lipopolysaccharide (LPS) is sensed by caspase 11 (caspase 4 in humans) which triggers self-cleavage and cleavage and activation of the pore forming protein, gasdermin D (GSDMD), followed by NINJ1 activation and plasma membrane rupture via a process termed pyroptosis ( 1 – 3 ). Potassium efflux induced by GSDMD pores indirectly triggers non-canonical NLRP3 and the maturation and release of IL-1β and IL-18 ( 4 ). Bacterial derived LPS can be released into circulation via the complement system, antibiotics and antimicrobial peptides ( 5 ). Additionally, free LPS can associate with extracellular vesicles (EVs) in vivo to gain access to the cytosol via CD14 ( 5 , 6 ). However, how this is controlled to maintain homeostasis is unknown. To identify previously unknown LPS sensors we performed a proteomic screen using biotinylated LPS. THP1 derived human macrophages were incubated with biotin-LPS followed by enrichment of LPS binding proteins using streptavidin beads. Mass spectrometry analysis identified M acrophage a ssociated r eceptor with co llagenous structure (MARCO) as an LPS binding protein ( Fig.1A-C ). MARCO is a scavenger receptor uniquely expressed on macrophages. MARCO has been implicated in the clearance of pathogens and apoptotic debris, tumorigenesis and autoimmunity ( 7 – 12 ). MARCO contains a scavenger receptor cysteine rich (SRCR) domain which is required for ligand binding ( 10 ). However, MARCO contains no signaling domain. Despite, these findings there is a lack of genetic studies and a paucity in our understanding of the functional role of MARCO plays in innate immunity and how MARCO expression is regulated on tissue resident macrophages. We next evaluated the expression level of MARCO in primary murine bone marrow derived macrophages (BMDMs). However, MARCO was not expressed at a protein level in BMDMs. We thus evaluated expression in non-differentiated and differentiated THP1 cells. PMA treatment results in the induction of MARCO in THP1 derived macrophages ( Fig.1D ) . We next evaluated if inflammatory signals could stimulate MARCO expression in BMDMs. The TLR2 ligand Pam3CSK4 induced robust MARCO expression. However, the TLR4 or TLR3 agonist LPS or Poly (I:C) failed to induce MARCO ( Fig.1E ) . Given that TLR2 signaling does not induce type I IFN signaling via TBK1 and IRF3 we hypothesized that autocrine IFN signaling restricted MARCO expression. Indeed, treatment with the JAK1 inhibitor ruxolitinib restored MARCO expression in LPS and Poly (I:C) treated macrophages. STING activation using diABZI failed to induce MARCO expression suggesting that TLR signaling is required for MARCO expression on macrophages ( Fig.1F -G ). We next re-evaluated LPS binding to MARCO in LPS primed BMDMs in the presence of ruxolitinib. Ruxolitinib treatment resulted in the precipitation of MARCO by biotin-LPS ( Fig.1H-J ). Biotin-LPS bound to recombinant MARCO by ELISA ( Fig.1K ) and colocalized with MARCO in Pam3CSK4 primed BMDMs ( Fig.1L ). FITC-labelled LPS also co-localized with MARCO ( Extended Fig.1A ) .Thus, MARCO is a previously unknown LPS binding protein controlled by IFN. Download figure Open in new tab Extended Figure 1: RNA sequencing analysis of ruxolitinib treated macrophages. (A) Immunofluorescence staining of MARCO and FITC-labelled LPS in WT BMDMs primed with Pam3CSK4 for 24 hours followed by incubation with FITC-LPS (500ng/ml) for 1 hour. (B) Heat map of differentially expressed genes from RNA-seq analysis of vehicle or ruxolitinib and LPS treated. (C) Top 20 most significantly upregulated genes in LPS and ruxolitinib treated cells relative to LPS treated cells. ( D-E) FPKM values of Marco (D) and Cxcl10 (E). A-D average of two-independent experiments from RNA sequencing. Download figure Open in new tab Figure 1: MARCO is an LPS binding protein. (A-B) Peptide map and relative abundance of MARCO in eluted from streptavidin beads incubated in PMA differentiated THP1 monocytes treated with PBS or biotin-LPS (500ng/ml) for 1 h. (C) Immunoblot analysis of MARCO from streptavidin elution from A. (D) Immunoblot analysis of MARCO and GAPDH in THP1 cells treated with or without PMA (50ng/ml) for 48 hours. (E) Immunoblot analysis of MARCO and GAPDH in WT BMDMs with treated with Pam3CSK4 (100ng/ml), LPS (100ng/ml) or Poly (I:C) (10ug/ml) for 24 hours. (F) RT-qPCR analysis of Marco mRNA in WT BMDMs treated with Pam3CSK4 (100ng/ml), LPS (100ng/ml), Poly (I:C) (10ug/ml), CpG DNA (1ug/ml) or diABZI (100nM) for 24 hours. (G) Immunoblot analysis of MARCO and GAPDH in WT BMDMs with the indicated ligands for 24 hours. (H-I) Peptide map and relative abundance of MARCO in eluted from streptavidin beads incubated in WT BMDMs pre-treated with ruxolitinib (100nM) and LPS for 24 hours followed by treatment with PBS or biotin-LPS (500ng/ml) for 1 h. (J) Immunoblot analysis of MARCO from streptavidin elution and input from H. (K) ELISA assay of immobilized recombinant MARCO incubated with biotin LPS. (L) Immunofluorescence staining of MARCO and biotin-labelled LPS in WT BMDMs primed with Pam3CSK4 for 24 hours followed by incubation with biotin-LPS for 1 hour. B, F, H, I pooled data from 3 independent experiments. C-E, G, J, L representative images from 3 independent experiments. *P<0.05, **P<0.01, student’s t-test. We next further assessed the ability of IFN to restrict MARCO expression. Using an unbiased approach, we sequenced LPS treated WT and Ifnar -/- BMDMs. Interestingly, MARCO was one of the most upregulated genes in LPS-treated Ifnar -deficient cells when compared to LPS treated WT cells ( Fig.2A-C ) . Expression of the IFN-stimulated genes (ISG), Cxcl10 , was lost in Ifnar -/- BMDMs ( Fig.2D ). Sequencing of LPS and ruxolitinib treated cells also identified MARCO as one of the most upregulated genes in ruxolitinib treated cells ( Extended Fig.1B-E ). Pre-treatment of cells with anti-IFNAR blocking antibody ( Fig.2E ), ruxolitinib or tofacitinib ( Fig.2F ) and Irf3 -/- or Ifnar -/- cells ( Fig.2E-H , Extended Fig. 2A ) all resulted in robust LPS-induced MARCO expression. Indirect stimulation of IFNβ via addition of exogenous recombinant IFNβ to Irf3 -/- cells restricted augmented MARCO ( Extended Fig.2B ). Additionally, exogenous IFNβ treatment suppressed Pam3CSK4 induced MARCO expression ( Extended Fig.2C ) . Flow cytometry analysis also demonstrated complete inhibition of TLR2 induced MARCO expression in the presence of type I (IFNβ) or type II (IFNγ) IFN ( Fig.2I-L ). Addition of exogenous IFNα, β or γ all suppressed TLR2 induced transcriptional activation and protein levels of MARCO ( Fig.2M-N ). Exogenous IFNγ suppressed TLR2 induced MARCO in both WT and Ifnar -/- cells ( Fig.2O ) . Collectively these data demonstrate that MARCO is an IFN-restricted gene. Download figure Open in new tab Extended Figure 2: IFN-negatively regulates MARCO expression. (A) Immunofluorescence staining of MARCO and Hoechst in WT and Marco -/- BMDMs treated LPS (100ng/ml) for 1 hour followed by ruxolitinib or tofacitinib (100nM) for 24 hours. (B) Immunoblot of MARCO and β-actin in WT and Irf3 -/- BMDMs treated LPS (100ng/ml) for 1 hour followed by IFNβ (10ng/ml) for 24 hours. (C) Immunofluorescence staining of MARCO and Hoechst in WT BMDMs treated with Pam3CSK4 (100ng/ml) for 1 hour followed by IFNβ (50ng/ml) for 24 hours. A-D representative images from 3 independent experiments. Download figure Open in new tab Figure 2: MARCO is an IFN-restricted gene. (A) Heat map of differentially expressed genes from RNA-seq analysis of WT and Ifnar -/- BMDMs treated with LPS for 6 hours. (B) Top 20 most significantly upregulated genes in LPS treated Ifnar -/- cells relative to WT LPS treated cells. ( C-D) FPKM values of Marco (C) and Cxcl10 (D). (E) Immunoblot analysis of MARCO and β-actin in WT BMDMs treated with LPS (100ng/ml) for 1 hour followed by anti-IFNAR (10ng/ml) for 24 hours. (F) Immunoblot analysis of MARCO and GAPDH in WT BMDMs treated with LPS (100ng/ml) for 1 hour followed by ruxolitinib or tofacitinib (100nM) for 24 hours. (G) Immunoblot analysis of MARCO and β-actin in WT, Ifnar -/- and Irf3 -/- BMDMs treated with LPS (100ng/ml) for 24 hours. (H) Immunofluorescence staining of MARCO and Hoechst in WT and Ifnar -/- BMDMs primed treated with LPS (100ng/ml) for 24 hours. (I-J) Representative flow cytometry plots (I) and quantification (J) of CD45+, F4/80 and MARCO positive cells treated with Pam3CSK4 (500ng/ml) for 1 hour followed by IFNβ (10ng/ml) for 24 hours. (K-L) Representative flow cytometry plots (K) and quantification (L) of CD45+, F4/80 and MARCO positive cells treated with Pam3CSK4 (500ng/ml) for 1 hour followed by IFNγ (10ng/ml) for 24 hours. (M) RT-qPCR analysis of Marco mRNA in WT BMDMs treated with Pam3CSK4 (100ng/ml) for 1 hour followed by treatment with IFNα (10ng/ml), IFNα (10ng/ml), IFNγ (10ng/ml) and IFNλ (10ng/ml) for 5 hours. (N) Immunoblot analysis of MARCO and GAPDH in WT and Marco -/- BMDMs treated with Pam3CSK4 (100ng/ml) for 1 hour followed by treatment with IFNα (10ng/ml), IFNβ (10ng/ml), IFNγ (10ng/ml) and IFNλ (10ng/ml). (O) Immunoblot analysis of MARCO and GAPDH in WT and Ifnar -/- BMDMs treated with Pam3CSK4 (100ng/ml) for 1 hour followed by IFNγ (10ng/ml) for 24 hours. A-D average of two-independent experiments. J, L, M pooled biological replicates from 3 independent experiments. E-H, I, K, N-O representative images from 3 independent experiments. **P<0.01, ***P<0.001student’s t-test. Given the limited literature on transcription factors that promote MARCO expression we performed a bioinformatic analysis on transcription factor binding sites in open chromatin regions (OCRs) of the MARCO locus. Analysis of ImmGen databases revealed that MARCO expression is limited to the macrophage lineage. Further, OCR analysis under low and high stringency models identified 4 putative MARCO transcription factors, JunD, Nfe2l2 ( NRF2 ), cFos and Smarcc1 ( Fig.3A-C ) . Given that the NRF2-dependent gene Hmox 1 was also elevated in Ifnar -deficient or ruxolitinib treated cells ( Extended Fig.3A-B ) we evaluated the requirement of NRF2 for MARCO expression. NRF2 is a transcription factor that responds to oxidative stress and induces antioxidant and metabolic gene expression ( 13 ). Under homeostatic conditions the E3 ligase KEAP1 constitutively degrades NRF2 via K48-linked ubiquitination and proteasomal degradation ( 14 , 15 ). Exposure of macrophages to electrophilic or oxidative stress results in the inhibition of KEAP1 and the stabilization of NRF2 ( 14 , 16 ). Deletion of NRF2 resulted in the complete loss of MARCO at a transcriptional and protein level ( Fig.3D-G , Extended Fig.3C ). The NRF2 kinase ERK was also required for MARCO expression ( Extended Fig.3D-E ) . Thus, NRF2 is required for MARCO expression. Given that IFN exposure potently suppresses MARCO expression we next evaluated the effect of IFN exposure on NRF2 stabilization. Remarkably, we failed to observe stabilization of NRF2 in LPS treated WT cells. However, Ifnar -deficient cells displayed robust stabilization and nuclear localization of NRF2 ( Fig.3H-I ). In contrast to other studies this suggests that autocrine IFN produced downstream of LPS stimulation creates a barrier that limits NRF2 dependent gene expression. TLR2-stimulation, which does not induce any IFN, induced a significant increase in NRF2 stabilization ( Fig.3J ). Furthermore, exogenous IFNβ treatment impaired Pam3CSK4 induced NRF2 stabilization ( Fig.3K-L ). We next evaluated the mechanism by which NRF2 is stabilized downstream of TLR2. Previous studies have demonstrated that metabolic changes downstream of TLR4 activation results in the expression of the enzyme Irg1 ( Acod1 ) which synthesizes the immunomodulatory metabolite itaconate ( 17 ). Itaconate and its derivative 4-octyl itaconate (4-OI) have been shown to promote NRF2 stabilization via different mechanisms ( 18 ). Indeed, 4-OI can directly alkylate KEAP1 to inhibit NRF2 degradation and NLRP3 to limit the NLRP3 inflammasome ( 14 , 19 ). Endogenous itaconate can also induce ROS production via the inhibition of succinate dehydrogenase (SDH)( 18 , 20 , 21 ). Itaconate can also modify GSDMD via itaconation to mediate macrophage tolerance ( 22 ). Nitric oxide (NO) has also been implicated in the stabilization of NRF2 via the inhibition of KEAP1( 23 ). Pam3CSK4 stimulation induced robust expression of Irg1 ( Fig.3M-N ). Further, TLR2-induced MARCO expression was significantly decreased in Irg1 -deficient BMDMs ( Fig.3O-P ). MARCO expression was also lost in Irg 1-deficient cells treated with LPS and ruxolitinib ( Fig.3Q-S ) . Although previous studies have indicated that Irg1 is an ISG, Irg1 expression was comparable between WT and Ifnar -/- cells ( Fig.3T ). Expression of Irg1 was also independent of NRF2 ( Fig.3U ). NO is synthesized by the ISG iNOS ( Nos2 ). Indeed, Nos2 gene expression was completely lost in Ifnar -/- cells ( Extended Fig.4A ). LPS induced NO synthesis in a Nos2 -dependent manner. TLR2 stimulation did not result in NO synthesis ( Extended Fig.4B ) . In line with previous studies NO synthesis was elevated in Irg1 -/- cells ( 24 ) ( Extended Fig. 4C ) . Treatment with the NO inhibitor SEIT suppressed LPS induced NO synthesis in WT and Irg1 -/- BMDMS ( Extended Fig. 4D-E ) . Ruxolitinib treatment inhibited LPS induced NO synthesis in WT and Irg1 -/- BMDMS ( Extended Fig. 4F-G ) due to suppression of the ISG iNOS ( Extended Fig.4H ) . SEIT treatment did not impair TLR2-induced MARCO expression ( Extended Fig.4I-J ) . Given that itaconate mediated inhibition of (SDH) can stimulate NRF2 activation via ROS production we hypothesized that MARCO expression was ROS dependent. Indeed, pre-treatment of cells with the ROS scavenger mitotempo suppressed MARCO expression ( Fig.3V -W) . These data demonstrate that MARCO expression and NRF2 activation can occur in an NO-independent and itaconate/ROS dependent manner. Thus, macrophages can only stabilize NRF2 in the absence of IFN through a NO-redundant mechanism downstream of TLR2. This process is then impaired when the macrophage is exposed to IFN. Download figure Open in new tab Extended Figure 3: IFN-negatively regulates NRF2 dependent gene expression. (A-B) FPKM values of Hmox1 LPS (100ng/ml) and ruxolitinib (100nM) treated cells (A) and LPS treated WT and Ifnar -/- BMDMs treated with LPS for 6 hours (B). (C) Immunofluorescence staining of MARCO and Hoechst in WT and Nfe2l2 -/- BMDMs treated Pam3CSK4 (100ng/ml) for 24 hours. (D) RT-qPCR analysis of Marco mRNA in WT BMDMs treated with or without AZ0364 (ERKi) for 1 hour followed by treatment with LPS (100ng/ml) and ruxolitinib (100nM) for 6 hours. (E) Immunoblot of MARCO and β-actin in lysates from WT BMDMs treated with or without AZ0364 (ERKi) 100nM for 1 hour followed by treatment with LPS (100ng/ml) and ruxolitinib (100nM) for 6 hours. A-B average of two-independent experiments from RNA sequencing described in Figure 2 and Extended Figure 1. C, E representative images from 3 independent experiments. D, pooled biological replicates from 3 independent experiments. Download figure Open in new tab Extended Figure 4: MARCO and NRF2 stabilization occur independently of nitric oxide. (A) FPKM values of Nos2 from RNA sequencing analysis of WT and Ifnar -/- BMDMs treated with or without LPS for 6 hours. (B-C) Nitric oxide assay on supernatants from WT and Nos2 -/- (B) or WT and Irg1 -/- (C) treated with Pam3CSK4 or LPS for 24 hours. (D-E) Nitric oxide assay on supernatants from WT and Nos2 -/- (D) or WT and Irg1 -/- (E) treated with LPS with or without SEIT (250mM) for 24 hours. ( F-G) Nitric oxide assay on supernatants from WT and Nos2 -/- (F) or WT and Irg1 -/- (G) treated with LPS with or without ruxolitinib (1μM) for 24 hours. (H) Immunoblot of iNos and GAPDH in lysates from WT BMDMs treated with or without ruxolitinib (1μM) for 1 hour followed by treatment with LPS (100ng/ml) for 24 hours. (I) RT-qPCR analysis of Marco mRNA in WT and Marco -/- BMDMs treated with Pam3CSK4 (100ng/ml) with or without SEIT (250mM) for the indicated times. (J) Immunoblot of MARCO and GAPDH in lysates from WT BMDMs treated with or without SEIT (250mM) for 1 hour followed by treatment with Pam3CSK4(100ng/ml) for 24 hours. A-G, I pooled biological replicates from 3 independent experiments. H, J representative images. **P<0.01, ***P<0.001,****P<0.0001 student’s t-test. Download figure Open in new tab Figure 3: IFN-exposure inhibits itaconate mediated NRF2 stabilization in macrophages. (A-C) Open chromatin region binging sites in the Marco locus. Data from ImmGen . (D) RT-qPCR analysis of Marco mRNA in WT and Nfe2l2 -/- BMDMs treated with Pam3CSK4 (100ng/ml) for 6 hours. (E) Immunoblot analysis of MARCO and GAPDH in WT and Nfe2l2 -/- BMDMs treated with Pam3CSK4 (100ng/ml) for 24 hours. (F) RT-qPCR analysis of Marco mRNA in WT and Nfe2l2 -/- BMDMs treated with ruxolitinib (100nM) for 1 hour followed by LPS (100ng/ml) for 5 hours. (G) Immunoblot analysis of MARCO and GAPDH in WT and Nfe2l2 -/- BMDMs treated with ruxolitinib (100nM) for 1 hour followed by LPS (100ng/ml) for 24 hours. (H) Immunofluorescence staining of NRF2 (cyan) and Hoechst (red) in WT, Ifnar -/- and Nfe2l2 -/- BMDMs treated with LPS (100ng/ml) for the indicated times. (I) Immunoblot analysis of NRF2 and GAPDH in WT and Ifnar -/- BMDMs treated with LPS (100ng/ml) for the indicated times. (J) Immunoblot analysis of NRF2 and GAPDH in WT and Nfe2l2 -/- BMDMs treated with Pam3CSK4 (100ng/ml) for the indicated times. (K) Immunofluorescence staining of NRF2 and Hoechst in WT, BMDMs treated with Pam3CSK4 (100ng/ml) for 1 hour followed by treatment with or without IFNβ (10ng/ml) for 5 hours. (L) Immunoblot analysis of NRF2 and GAPDH in WT BMDMs pre-treated with IFNβ (10ng/ml) followed by treatment with Pam3CSK4 (100ng/ml) for the indicated times. (M) RT-qPCR analysis of Irg1 mRNA in WT and BMDMs treated with or without Pam3CSK4 (100ng/ml) for 6 hours. (N) Immunoblot analysis of IRG1 in WT and BMDMs treated with or without Pam3CSK4 (100ng/ml) for 24 hours. (O) Immunoblot analysis of MARCO, IRG1 and GAPDH in WT and BMDMs treated with or without Pam3CSK4 (100ng/ml) for 24 hours. (P) Densitometry analysis of MARCO normalized to GAPDH from (P). (Q) RT-qPCR analysis of Marco mRNA in WT and Irg1 -/- BMDMs treated with or without ruxolitinib (100nM) for 1 hour followed by LPS for 5 hours. (R) Immunoblot analysis of MARCO and GAPDH in WT and Irg1 -/- BMDMs treated with or without ruxolitinib (100nM) for 1 hour followed by LPS for 5 hours. (S) Densitometry analysis of MARCO normalized to GAPDH from (R). (T) FPKM values of Irg1 in RNA seq of WT and Ifnar -/- BMDMs treated with or without LPS for 6 hours. (U) Immunoblot analysis of IRG1 in WT and Nfe2l2 -/- BMDMs with Pam3CSK4 (100ng/ml) for 24 hours. (V) RT-qPCR analysis of Marco mRNA in WT and BMDMs treated with or without Pam3CSK4 (100ng/ml) and Mitotempo for 6 hours. (W) Immunoblot analysis of MARCO and GAPDH in lysates from WT BMDMs treated with or without Pam3CSK4 (100ng/ml) and mitotempo for 24 hours. D, F, M, Q, T, V pooled biological replicates from 3 independent experiments. E, G, I-J, L, N, O, P, R, S, U and X representative images from 3 independent experiments. **P<0.01, student’s t-test. The potent effect IFN has on NRF2 suggests a priming event required for optimal immune responses and silencing of the anti-inflammatory function of NRF2. We next evaluated the functional significance of MARCO binding LPS and how IFN exposure can counteract this. WT and Marco -/- BMDMs displayed comparable signal 1 responses with no changes in TLR4 induced gene expression or activation of TBK1 and IRF3 ( Extended Fig.5A-F ) . We next evaluated the canonical NLRP3 inflammasome. Marco -/- BMDMs displayed comparable levels of nigericin induced pyroptosis, GSDMD activation and LDH release ( Fig.4A -D ). These data are consistent with LPS priming failing to induce MARCO expression in BMDMs. We next assessed caspase 11 and non-canonical inflammasome responses in tissue resident macrophages by administering LPS to mice. MARCO was expressed in splenic macrophages ( Fig.4E ). Further, intraperitoneal delivery of LPS resulted in the loss of MARCO on splenocytes ( Fig.4F ). Splenic expression of MARCO was dependent on NRF2 ( Fig.4G ). WT and Marco -deficient mice displayed a comparable immune cell profile in the spleen ( Extended Fig.6A-K ) . Caspase 11 and GSDMD are essential for LPS induced septic shock. Pre-treatment of mice with an anti-MARCO antibody result in enhanced LPS-induced septic shock ( Fig.4H ) . Marco -/- mice also displayed enhanced sensitivity to LPS-shock and serum levels of IL-1β ( Fig.4I -L ). Neutralization of MARCO also enhanced LPS sensitivity in Tlr4 -/- mice ( Fig.4M ) . Nfe2l2 -/- , Irg1 -/- or administration of the KEAP1 activator VVD133037 all enhanced lethality in response to high dose LPS ( Extended Fig.7A-C ). In line with previous studies Ifnar -/- were protected against LPS-shock ( Extended Fig.7D ). We next evaluated caspase 11 activation. Marco -deficient mice displayed enhanced caspase 11 activation when compared to WT mice ( Fig.4N ). Caspase 11 is an ISG and is upregulated on macrophages following IFN exposure as a priming event that precedes non-canonical inflammasome activation ( 25 ). Thus, IFN induced caspase 11 upregulation is accompanied by the concomitant downregulation of MARCO. Indeed, MARCO-expressing tissue resident macrophages downregulate MARCO following exposure to IFNβ ( Fig.4O ) . Although high dose LPS can be useful in studying septic shock responses it does not fully recapitulate the complexity of sepsis. Polymicrobial sepsis can be induced in mice via the IP administration of cecal slurry. Cecal slurry via IP mimics the symptoms of sepsis without using invasive surgical procedures such as cecal ligation puncture (CLP). Marco -/- mice subjected to polymicrobial sepsis displayed enhanced lethality when compared to WT mice ( Fig.4P ). Download figure Open in new tab Extended Figure 5: MARCO is dispensable for TLR4 signaling in BMDMs. (A-E) RT-qPCR analysis of Il1 β , Il6, Cxl10, Ifit1, Ifit2 mRNA in WT and Marco -/- BMDMs treated with LPS (100ng/ml) for the indicated times. (F) Immunoblot analysis of phosphorylated IRF3 and TBK1 in lysates from WT and Marco -/- BMDMs treated with LPS (100ng/ml) for the indicated times. A-E pooled biological replicates from 3 independent experiments. F, representative images from 3 independent experiments. Download figure Open in new tab Extended Figure 6: Splenic immune cell profile of Marco -deficient mice. (A) Representative flow plots and gating strategy of live, single, CD45+ cells. (B-C) Spleen weight (B) and splenocyte number (C) of spleens from WT and Marco -/- mice. (D-K) Cell counts of the indicated cell type in spleens from WT and Marco -/- mice. n =5-9 mice per group. Download figure Open in new tab Extended Figure 7: Loss of NRF2 or itaconate sensitizes mice to LPS shock. (A) Survival analysis of WT and Nfe2l2 -/- mice administered LPS (40mg/kg). (B) Survival analysis of WT and Irg1 -/- mice administered LPS (40mg/kg). (C) Survival analysis of WT mice administered LPS (40mg/kg) with or without pre-treatment with VVD133037 (10mg/kg). (D) Survival analysis of WT and Ifnar -/- mice administered LPS (40mg/kg). **P<0.01, Mantel-Cox analysis. Download figure Open in new tab Figure 4: IFNs prime the non-canonical inflammasome through inhibition of MARCO. (A-B) Representative image (A) and quantification (B) of SYTOX orange positive cells of assay of SYTOX orange and Hoechst-stained WT and Marco -/- BMDMs treated with LPS (100ng/ml) for 3 hours and nigericin (10μM) for 1 hour. (C) LDH assay on supernatants from WT and Marco -/- BMDMs treated with LPS (100ng/ml) for 3 hours and nigericin (10μM) for 1 hour. (D) Immunoblot analysis of GSDMD and GAPDH in lysates from WT and Marco -/- BMDMs treated with LPS (100ng/ml) for 3 hours and nigericin (10μM) for the indicated times. (E) Immunofluorescence staining of MARCO F4/80 and Hoechst from splenic tissue section from WT and Marco -/- mice. (F) Immunoblot of MARCO and GAPDH on lysates from digested spleens from mice injected IP with PBS or LPS (10mg/kg) for 6 hours. (G) Immunoblot of MARCO and GAPDH on lysates from digested spleens from WT and Nfe2l2 -/- mice. (H ) Survival analysis of WT mice administered anti-MARCO antibody (50mg/kg) for 1 hour followed by LPS (40mg/kg). (I) Survival analysis of WT and Marco -/- administered LPS (40mg/kg). (J-K) Survival analysis of WT and Marco -/- administered the indicated concentrations of LPS. (L) ELISA analysis of IL-1β on serum from WT and Marco -/- administered LPS (10mg/kg) for 6 hours. (M) ELISA analysis of IL-1β on serum from WT and Marco -/- administered LPS (10mg/kg) for 5 hours. (M) Survival analysis of Tlr4 -/- mice administered LPS (40mg/kg) with or without anti-MARCO antibody (50mg/kg). (N) Immunoblot of caspase 11, and GAPDH on lysates from digested spleens from WT and Marco -/- administered LPS (10mg/kg) for 5 hours. (O) Immunoblot of MARCO and GAPDH in lysates from peritoneal macrophages treated with IFNβ (50ng/ml) for 24 hours. (P) Survival analysis of WT and Marco -/- administered 200 μl cecal slurry. B, C pooled biological replicates from 3 independent experiments. F-G, n =3 mice per group. D, O representative images from 3 independent experiments. H-M, P. n =3-10 mice per group. *P<0.05**P<0.01,****P<0.0001 student’s t-test or Mantel-cox survival analysis. These data uncover a previously unknown feedback loop whereby IFN exposure downregulates MARCO in tissue resident macrophages or suppresses the upregulation of MARCO in BMDMs to maximize the detection of cytosolic LPS. Further, we identify the mechanism by which IFN-restricts MARCO expression via the inhibition of itaconate mediated NRF2 stabilization. Our study also shows for the first time that NRF2 stabilization can occur in the absence of NO but with a requirement for itaconate and ROS. This explains why NRF2 stabilization is not observed in macrophages in response to LPS. Our study uncovers the first known metabolic LPS sensor and a previously unknown regulatory mechanism that controls NRF2 activity in macrophages. Further understanding the role MARCO plays in restricting LPS sensing and the signaling events that govern its expression is likely to yield new insights into the pathogenesis and treatment of sepsis and other inflammatory diseases. Materials and Methods Mice Marco -/- mice were generated by Taconic. Irg1 -/- , Nrf2 -/- , Nos2 -/- , Tlr4 -/ were obtained from Jackson laboratories. All animal experiments were approved by the Institutional Animal Care Use Committees at the University of Massachusetts Medical School. Animal were kept in SPF environment. Immunoblotting and Immunoprecipitation Primary BMDMs from mice were cultured in 12-well plates (1×10 6 cells per ml; 1 ml) or 10-cm dishes (2×10 6 cells per ml; 10ml). For cell lysate analysis cells were lysed directly in 1X Lamelli sample buffer. For native gel analysis cells were lysed in in NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 0.5% (w/v) IgePal, 50 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and protease 4 inhibitor cocktail. For immunoprecipitation of GSDMD, cells were treated as indicated and then collected in 500 µl RIPA buffer, followed by incubation for 15 min on ice. Lysates were incubated with GSDMD antibody and protein A–protein G-agarose was added to each sample, followed by incubation overnight at 4°C. Immunoprecipitates were collected by centrifugation and washed four times with 1 ml of RIPA buffer. Immunprecipates were eluted from beads using 1X sample buffer. Samples were resolved by SDS-PAGE and stained using simply blue safe stain or transferred to nitrocellulose membranes and analyzed by immunoblot. Immunoreactivity was visualized by the Odyssey Imaging System (LICOR Biosciences). For immunoblotting of IL-1β in cell supernatants, conditioned medium was collected and filtered using filter spin columns to reduce salt and remove abundant serum proteins. Filtrates were added to 4X SDS-PAGE sample buffer and resolved by SDS-PAGE for immunoblot analysis. GSDMD (ab209845) were from abcam. anti-mouse IRDyeTM 680 (926–68070) and anti-rabbit IRDyeTM 800 (926–32211) were from LI-COR Biosciences. Cells were lysed directly in 1x Laemmli Sample Buffer (Bio Rad) containing β- Mercaptoethanol (Sigma) and heated at 95°C for 10 minutes. Samples were resolved using SDS-PAGE and transferred to nitrocellulose membranes. Samples were immunoblotted with the indicated antibodies. Proteins were detected using fluorophore-conjugated anti-rabbit secondary antibody (LICOR Biosciences) and immunoreactivity was visualized using the Odyssey Imaging System (LICOR Biosciences). RT-qPCR Cells were lysed in TRIzol reagent (Invitrogen), and total RNA was isolated using the Direct-zol RNA miniprep kit (Zymo Research). RNA was quantified by a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and 1ug of RNA was reverse transcribed using iScript Reverse Transcription Supermix (Bio Rad). qPCR analysis was done on 20 ng of cDNA using the iQ SYBR Green super-mix reagent (Bio Rad). Gene expression was normalized to TATA-binding protein (TBP) expression and relative mRNA expression was calculated by a change in cycling threshold method as 2 -ΔΔC(t) . Amplification specificity was assessed via melting curve analysis. Kinetic cell death analysis BMDMs were seeded (0.1 x 10 6 cells per well) in RPMI-1640 media (ATCC) supplemented with 10% FBS, 1% penicillin and streptomycin and 5% L929 conditioned media in a 96 well plate and rested overnight. To activate the canonical inflammasome, cells were primed with 100 ng/mL LPS (Sigma) for 3 hours and stimulated with 5 μM nigericin (Cayman Chemical). To activate the noncanonical inflammasome, cells were primed with 1 μg/μl Pam3CSK4 (Invitrogen) for 8 hours and transfected with 1 μg LPS (Sigma) using Lipofectamine 3000 (Invitrogen). Cells were stained with 0.25 μM SYTOX orange (Thermo Scientific) and 1 ug/mL Hoechst (Thermo Scientific) and visualized using the BioTek Lionheart FX Microscope (Agilent). The ratio of SYTOX positive cells to Hoechst positive cells were quantified using the BioTek Gen5 Software. L929 conditioned media L929 cells were seeded in T175 flask in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin. Cells were rested for 10 days after reaching confluency at 37 °C in a humidified atmosphere of 5% CO 2 . Conditioned media was passed through a 0.2 um filter before use. Bone marrow derived macrophage culture Femur and tibia bones were removed from 8-12-week-old mice and bone marrow was flushed using fresh RPMI media. Bone marrow was cultured in RPMI-1640 media (ATCC) supplemented with 10% FBS, 1% penicillin and streptomycin, and 20% L929 conditioned media produced as described above. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 7 days. Flow cytometry Flow cytometry was performed as previously described ( 26 ). Briefly, BMDMs were stained in MACS buffer (0.5% BSA, 2 mM EDTA) using Ghost Violet 540 viability dye (TONBO), anti-CD45.2 BV650 (BioLegend), CD11c PE (BioLegend), and MARCO APC (eBioscience). Cells were acquired on a CytekTM Aurora. Flow cytometry analysis was done using FlowJo software. RNA sequencing RNA sequencing was performed by BGI as previously described( 27 ). Peptide mapping by nano LC-MS/MS Streptavidin pull downs were eluted in pierce elution buffer and subjected to in solution to in-gel digestion with trypsin. The resulting peptides were lyophilized, re-suspended in 5% acetonitrile, 0.1% (v/v) formic acid in water and injected onto a NanoAcquity UPLC (Waters) coupled to a Q Exactive (Thermo Scientific) hybrid quadrapole orbitrap mass spectrometer. Peptides were trapped on a 100 µm I.D. fused silica pre-column packed with 2 cm of 5 µm (200Å) Magic C18AQ (Bruker-Michrom) particles in 5% acetonitrile, 0.1% (v/v) formic acid in water at 4.0 µl/min for 4.0 minutes. Peptides were then separated over a 75 µm I.D. gravity-pulled 25 cm long analytical column packed with 3 µm (100Å) Magic C18AQ particles, at a flow rate of 300 nl/min containing mobile phase A, 0.1% (v/v) formic acid in water and mobile phase B, 0.1% (v/v) formic acid in acetonitrile, using a biphasic gradient: 0-60 min (5-35% B), 60-90 min (35-60% B), 90-93 min (60% B), 93-94 min (60-90% B), 94-109 (90% B), followed by equilibration to 5% B. Nano-ESI source was operated at 1.4 kV via liquid junction. Mass spectra were acquired over m/z 300-1750 at 70,000 resolution (m/z 200) with an AGC target of 1e6. Data-dependent acquisition (DDA) selected the top 10 most abundant precursor ions for tandem mass spectrometry by HCD fragmentation using an isolation width of 1.6 Da, max fill time of 110ms, and AGC target of 1e5. Peptides were fragmented by a normalized collisional energy of 27, and product ion spectra were acquired at a resolution of 17,500 (m/z 200). Raw data files were peak processed with Proteome Discoverer (version 2.1, Thermo Scientific) followed by identification using Mascot Server (Matrix Science) against the Mouse (Swissprot) FASTA file (downloaded 07/2019). Search parameters included full tryptic enzyme specificity, and variable modifications of N-terminal protein acetylation, oxidized methionine, glutamine conversion to glutamic acid. Assignments were made using a 10-ppm mass tolerance for the precursor and 0.05 Da mass tolerance for the fragment ions. All non-filtered search results were processed by Scaffold (version 4.8.4, Proteome Software, Portland, OR) utilizing the Trans-Proteomic Pipeline (Institute for Systems Biology, Seattle, WA) at 1% false-discovery rate (FDR) for peptides and 99% threshold for proteins (2 peptides minimum). Immunofluorescence Coverslips were handled with sterile forceps, retrieved from −20°C storage, and placed in 24-well plates. BMDM cells were seeded at 200,000 cells per well in 0.5 mL media and incubated overnight. Treatments were applied directly to the wells the following day, and staining was performed in the same plates. Media was removed, and cells were washed with 500 µL PBS. Fixation was carried out with 200 µL of 4% paraformaldehyde for 10 minutes, followed by three PBS washes. Cells were permeabilized with 0.1% Tween-20 for 10 minutes and washed three times. Blocking was done using 200 µL of 5% normal goat serum for 45 minutes. Primary antibodies (1:200 dilution) were added dropwise (50 µL) to coverslips and incubated for 1 hour at room temperature. After four PBS washes, secondary antibodies (1:200) were applied (50 µL), incubated for 1 hour, and washed four times. Coverslips were carefully removed using a bent needle. Slides were prepared with a drop of DAPI-containing mounting solution. Coverslips were placed on the solution after excess liquid was blotted, and slides were cured overnight before imaging. Survival Analysis LPS (Sigma L2630-100MG) was dissolved in water at 5mg/ml. The mice were intraperitoneally injected with 40mg/kg of LPS or equivalent volume of Vehicle (water). Survival was then monitored over time. Genotyping Ear clips from the mouse tail were incubated with 50nM NaOH for 1 hour at 55 degrees. The DNA digest was then used for PCR to detect WT and KO alleles. Histology (spleen tissue) Immunofluorescence staining of mouse spleens was performed by Applied Pathology Systems, LLC. Formalin-Fixed Paraffin Embedded (FFPE): spleen tissues were sectioned and mounted to glass slides. Tissue sections were dewaxed, rehydrated, and subjected to the antigen retrieval using 2100 antigen retriever (Aptum Biologics Ltd). Fluorescence co-stained slides were cover slipped with Fluro mount-G Mounting media (Thermo) before imaging with Leica Thunder microscope. Briefly, all the tissues were fixed in 10% formalin (Sigma: MKCS 3994) for 24 hours and then transferred to 70% ethanol. The fixed tissues undergo dehydration through a series of graded alcohols, clearing with xylene to remove alcohol prior to paraffin infiltration and embedding. Thin tissue sections, typically 4–5 µm thick, are sectioned with microtome and mounted on glass slides and dried in an oven at 60°C for 1 hour before H&E staining. Antigen retrieval was performed on dewaxed and rehydrated FFPE sections using R-Buffer A (Fisher Scientific, 50311711; immunofluorescence) in a pressure cooker. For immunohistochemistry staining, the tissue sections were blocked with 5% donkey serum in 0.3% Triton-X in PBS. The tissue sections were incubated with primary antibodies for 1 hours and washed 3 times with PBS tween (0.1%), followed by incubation with secondary antibodies for 1 hour at RT. Primary antibody was made in blocking buffer and secondary antibodies were made in PBS. The slides were washed 3 times with PBS and mounted using DAPI containing Fluro mount-G Mounting media. Statistical analysis For comparisons of two groups two-tailed students’ t test was performed. Multiple comparison analysis was performed using two-way ANOVA. Mann-Whitney U test was used for EAE analysis. Mantel-Cox were used for survival analysis. Three to ten mice were used per experiment, sufficient to calculate statistical significance, and in line with similar studies published in the literature. Ethics All animal studies were performed in compliance with the federal regulations set forth in the Animal Welfare Act (AWA), the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and the guidelines of the UMass Medical School Institutional Animal Use and Care Committee. All protocols used in this study were approved by the Institutional Animal Care and Use Committee at the UMass Medical School (protocol 202200082). Author Contributions SR and SC performed experiments and analyzed data. MF assisted with animal experiments and animal husbandry. LS assisted with experiments. LG performed experiments and analyzed data and edited the manuscript. FH conceived the study, developed the concept, performed experiments, analyzed data and wrote the manuscript Acknowledgements FH is funded by start-up funding from UMass Chan Medical School, Charles H.Hood foundation, Riccio Fund for neuroscience and the UMass Chan BRIDGE fund. References 1. ↵ N. Kayagaki et al. , Non-canonical inflammasome activation targets caspase-11 . Nature 479 , 117 – 121 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 2. N. Kayagaki et al. , Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling . Nature 526 , 666 ( 2015 ). 3. ↵ N. Kayagaki et al. , NINJ1 mediates plasma membrane rupture during lytic cell death . Nature 591 , 131 – 136 ( 2021 ). OpenUrl CrossRef PubMed 4. ↵ N. 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Share MARCO is an IFN-restricted immunometabolic decoy LPS receptor Shriram Ramani , Sara Cahill , Matthew Finnegan , Laurel Stine , Liraz Shmuel-Galia , Fiachra Humphries bioRxiv 2025.03.10.642106; doi: https://doi.org/10.1101/2025.03.10.642106 Share This Article: Copy Citation Tools MARCO is an IFN-restricted immunometabolic decoy LPS receptor Shriram Ramani , Sara Cahill , Matthew Finnegan , Laurel Stine , Liraz Shmuel-Galia , Fiachra Humphries bioRxiv 2025.03.10.642106; doi: https://doi.org/10.1101/2025.03.10.642106 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Immunology Subject Areas All Articles Animal Behavior and Cognition (7616) Biochemistry (17625) Bioengineering (13851) Bioinformatics (41824) Biophysics (21397) Cancer Biology (18524) Cell Biology (25417) Clinical Trials (138) Developmental Biology (13350) Ecology (19858) Epidemiology (2067) Evolutionary Biology (24277) Genetics (15580) Genomics (22459) Immunology (17698) Microbiology (40278) Molecular Biology (17134) Neuroscience (88400) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4812) Physiology (7632) Plant Biology (15106) Scientific Communication and Education (2042) Synthetic Biology (4281) Systems Biology (9807) Zoology (2266)